EP2628488B1 - Sustained-release pharmaceutical composition - Google Patents

Sustained-release pharmaceutical composition Download PDF

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Publication number
EP2628488B1
EP2628488B1 EP11832614.9A EP11832614A EP2628488B1 EP 2628488 B1 EP2628488 B1 EP 2628488B1 EP 11832614 A EP11832614 A EP 11832614A EP 2628488 B1 EP2628488 B1 EP 2628488B1
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EP
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Prior art keywords
collagen
sustained
sugar
pharmaceutical composition
drug
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EP11832614.9A
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German (de)
English (en)
French (fr)
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EP2628488A1 (en
EP2628488A4 (en
Inventor
Masanori Fukushima
Hiroaki Matsubara
Satoaki Matoba
Shigeki Hijikata
Yu Aso
Tsutomu Sato
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Advanced Innovation Development Co Ltd
Medical Research And Development Corp
Koken Co Ltd
Kyoto Prefectural PUC
Original Assignee
Advanced Innovation Development Co Ltd
Medical Research And Development Corp
Koken Co Ltd
Kyoto Prefectural PUC
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Publication of EP2628488A1 publication Critical patent/EP2628488A1/en
Publication of EP2628488A4 publication Critical patent/EP2628488A4/en
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0002—Galenical forms characterised by the drug release technique; Application systems commanded by energy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7004—Monosaccharides having only carbon, hydrogen and oxygen atoms
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7008—Compounds having an amino group directly attached to a carbon atom of the saccharide radical, e.g. D-galactosamine, ranimustine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1825—Fibroblast growth factor [FGF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/39—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A61K9/0024—Solid, semi-solid or solidifying implants, which are implanted or injected in body tissue
    • A—HUMAN NECESSITIES
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    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/08—Solutions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/5123—Organic compounds, e.g. fats, sugars
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/02—Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • A—HUMAN NECESSITIES
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    • A—HUMAN NECESSITIES
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1808—Epidermal growth factor [EGF] urogastrone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1816—Erythropoietin [EPO]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1858—Platelet-derived growth factor [PDGF]
    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/27—Growth hormone [GH], i.e. somatotropin

Definitions

  • the present invention relates to a sustained-release pharmaceutical composition and a method of manufacturing a sustained-release pharmaceutical composition.
  • a polymer material has been widely utilized in a sustained-release pharmaceutical composition because it allows a concentration of a drug in a tissue to be effectively retained over a long period.
  • the polymer material has such characteristics that the material can bind a drug in a physical or chemical manner and releases the drug along with progression of its in vivo degradation.
  • Such polymer material is exemplified by a cellulose derivative, a starch derivative, a dextran derivative, a polysaccharide, a protein, a polypeptide, an acrylic acid derivative, or a vinyl derivative.
  • Patent Literature 1 discloses that when the collagen concentration is reduced, degradation of a gelated collagen containing a drug is accelerated, sustained release of the drug is also accelerated, and thus a drug efficacy period is completed within a short period, and in contrast, that when the collagen concentration is increased, degradation of a gelated collagen is delayed, sustained release of the drug is also delayed, and thus drug efficacy can be exhibited for a long period.
  • Patent Literature 2 discloses a local sustained-release formulation for wound healing promotion obtained by mixing a carrier containing a collagen as an essential constituent with a physiologically active substance having a wound healing promoting activity.
  • the carrier described in Patent Literature 2 is constructed of ingredients selected from the group consisting of proteins such as a collagen and an albumin, carbohydrates such as a chitin, and synthetic polymers.
  • Patent Literature 2 discloses that a sustained-release characteristic of a drug depends on dosage forms such as a powder and a film. However, it takes much time to form the formulation into any of those dosage forms, and thus the formulation is lack of convenience.
  • Patent Literature 3 discloses a sustained-release formulation containing a collagen, a glycosaminoglycan as a polysaccharide, and a drug. Patent Literature 3 discloses that the glycosaminoglycan controls fibrillogenesis of the collagen and functions as a release control factor for the drug.
  • the sustained-release formulation of Patent Literature 3 is a solid formulation, is formed into a dosage form such as a needle or a stick, and hence is lack of convenience as with the formulation described in Patent Literature 2.
  • Patent Literature 4 discloses a composition comprising a collagen, a drug and either the disaccharide sucrose at a concentration of 0.03 M or the monosaccharide glucose at a concentration of 0.05 M, which is a lower sugar concentration than that of the composition of the invention.
  • Non Patent Literature 1 and Non Patent Literature 2 It is known that a collagen solution undergoes gelation when heated to around a body temperature in vitro, and it is also known that addition of a sugar such as a monosaccharide or a disaccharide to a collagen solution in advance inhibits gelation of the solution.
  • a sugar such as a monosaccharide or a disaccharide
  • Non Patent Literature 2 In a state in which the gelation is inhibited, it is considered that a sustained-release characteristic of a drug mediated by a collagen is not sufficiently exhibited.
  • a sustained-release pharmaceutical it has been impossible to employ a technology for inhibiting gelation using the collagen in combination with a sugar such as a monosaccharide.
  • An object of the present invention is to provide a pharmaceutical composition, including a drug and a collagen, in which the composition is satisfactory in handleability and has sustained-release property.
  • the present invention includes the following items.
  • sustained-release pharmaceutical composition of the present invention it is possible to achieve the controlled release of the drug as an active ingredient in an embodiment mode using immediately effective release and slow sustained release in combination in vivo.
  • immediately effective drug release can be attained by virtue of the fact that the addition of the sugar inhibits the gelation of the collagen in the initial period after the administration
  • slow sustained-release can be attained by virtue of the fact that the in vivo gelation of the collagen occurs in a time-dependent manner after the administration, resulting in a reduction in release rate of the drug.
  • the addition of the sugar to the collagen inhibits such gelation, which can facilitate a preparation operation at room temperature.
  • the present invention relates to a sustained-release pharmaceutical composition, including: a drug; a collagen; and at least one kind of sugar selected from disaccharides and trisaccharides, wherein the sugar has such a concentration in the composition before administration to a body that the sugar inhibits gelation of the collagen, wherein the concentration of the sugar is 0.1 M to 0.5 M.
  • the sustained-release pharmaceutical composition of the present invention is in a liquid form before in vivo administration, and underdoes gelation after in vivo administration.
  • the gelation of the sustained-release pharmaceutical composition of the present invention is inhibited by the presence of the sugar immediately after in vivo administration, but the inhibition of the gelation of the collagen by the sugar is cancelled as the sugar gradually diffuses in the living body, and the collagen is estimated to restore its intrinsic property and undergo gelation. That is, the gelation of the sustained-release pharmaceutical composition of the present invention slowly progresses after administration to a living body, and a drug sustained-release action can be finally exhibited.
  • the sustained-release pharmaceutical composition of the present invention is considered to have immediate-release property immediately after administration to a living body. As described above, the sustained-release pharmaceutical composition of the present invention has immediate-release property and sustained-release property, and has a controlled drug release rate.
  • the phrase "has immediate-release property" means that a pharmaceutical composition releases an effective amount of a drug in a period from immediately after administration to a given time (initial period).
  • the pharmaceutical composition of the present invention releases a drug at a rate comparable to or slightly slower than that of a composition containing no collagen, and at a rate faster than that of a composition containing a collagen only and containing nosugar.
  • the pharmaceutical composition of the present invention can release a drug in an amount sufficient to provide a subject with an initial loading dose of the drug within a time of 5 hours or less, 2 hours or less, 1 hour or less, or about 30 minutes or less.
  • the phrase "has sustained-release property" means that a pharmaceutical composition releases a drug in a sustained manner for a long period as compared to a composition containing no collagen to maintain the concentration of the drug in vivo at an appropriate one.
  • the pharmaceutical composition of the present invention can release a drug in an amount sufficient to provide a living body of a subject with a continuous maintenance dose of the drug for the following period: after a lapse of 12 hours or more, after a lapse of 16 hours or more, or after a lapse of 1 day or more after administration and up to about 3 days, up to about 1 week, or up to about 1 month after administration; or after a lapse of 1 month or more and up to about 6 months after administration.
  • a drug release rate can be more freely controlled by appropriately adjusting, for example, the concentrations, blending ratios, and blending order of ingredients including a drug, a collagen, and a sugar.
  • an initial drug release amount can be increased as compared to a conventional composition containing a collagen only and containing no sugar, and immediate-release property can be imparted.
  • a drug release rate can be regulated within such a range that a drug elution amount (cumulative drug release amount) 0 to 6 hours after the start of elution is increased by about 5 to 30% and a drug elution amount (cumulative drug release amount) 6 to 48 hours after the start of elution is increased by 30 to 50% as compared to a conventional composition containing a collagen only and containing no sugar.
  • each of those numerical values is an example of an initial drug release rate (a time range in which immediate-release property is exhibited and a cumulative drug release amount in the time range) in the sustained-release pharmaceutical composition of the present invention, and the initial drug release rate in the present invention is not limited thereto and can be controlled by the kinds and concentrations of a drug, a sugar, and a collagen.
  • the pharmaceutical composition of the present invention is in a liquid form before administration to a body and has property of undergoing gelation after the administration.
  • the gelation of the pharmaceutical composition of the present invention is probably due to a collagen. It is known that the gelation of the collagen generally occurs under physiological conditions.
  • the physiological conditions mean the conditions of a temperature of 2 to 45°C, a pH of 4.0 to 10.0, and a salt concentration of 0.01 to 3.0 M.
  • the pharmaceutical composition of the present invention is still in a liquid form and does not undergo gelation even when placed under physiological conditions before administered to a body.
  • the liquid form and gelated state in the present invention may be distinguished from each other by measuring a turbidity. For example, the turbidity may be confirmed by measuring an absorbance at 400 nm.
  • the property of undergoing gelation means having such property that an absorbance at 400 nm increases, for example, such property that the absorbance increases to about 1.0, when a composition containing a collagen is placed under physiological conditions.
  • the sugar contained in the sustained-release pharmaceutical composition of the present invention is at least one kind of sugar selected from disaccharides and trisaccharides.
  • the disaccharides include sucrose, maltose, lactose, trehalose, and isomaltose.
  • the trisaccharides include raffinose.
  • the sugar contained in the sustained-release pharmaceutical composition of the present invention is preferably a disaccharide.
  • a disaccharide and a trisaccharide are comprised from the viewpoint of handleability in the preparation of the pharmaceutical composition as well.
  • the sustained-release pharmaceutical composition when the sustained-release pharmaceutical composition is produced by allowing a collagen to coexist with polysaccharides including penta- or more saccharides in place of at least one kind of sugar selected from disaccharides and trisaccharides, it is considered that a drug sustained-release rate cannot be regulated because drug sustained-release property by the collagen is not substantially affected.
  • the concentration of the sugar in the sustained-release pharmaceutical composition of the present invention is one at which the sugar is capable of inhibiting gelation mediated by the collagen at a final concentration in the pharmaceutical composition, and is such a concentration that the pharmaceutical composition of the present invention does not undergo gelation even when placed under physiological conditions.
  • the concentration of the sugar in the pharmaceutical composition of the present invention may be increased or decreased depending on the addition of other ingredients in the pharmaceutical composition, and is preferably adjusted to an isotonic concentration with respect to an osmotic pressure in vivo as a whole.
  • the concentration of the sugar is 0.1 to 0.5 M. When the concentration of the sugar is less than 0.01 M, there is a problem in that a gelation inhibiting effect is not obtained. When the concentration is more than 3.0 M, there is a problem in that the gelation of the collagen is completely inhibited and the composition does not exhibit drug sustained-release property.
  • sustained-release pharmaceutical composition of the present invention by changing the kind and concentration of the sugar, it is possible to regulate a drug release rate and to regulate a balance between immediate-release property immediately after administration to a living body and sustained-release property due to the gelation of the collagen.
  • the balance between immediate-release property and sustained-release property may be regulated depending on, for example, the kind of the drug.
  • the sustained-release pharmaceutical composition of the present invention containing a disaccharide or a trisaccharide has a relatively high ability to suppress the gelation of the collagen, and hence can realize a higher local concentration through drug release in an initial period after administration, which enables immediately effective drug release.
  • a sustained-release pharmaceutical composition contains a monosaccharide or a tetrasaccharide
  • the composition has a relatively low ability to suppress the gelation of the collagen, and hence can reduce a drug release rate as compared to a formulation containing a disaccharide or a trisaccharide.
  • concentration of the sugar in the sustained-release pharmaceutical composition of the present invention is as described above, and is exemplified by the following concentrations: preferably 0.1 M to 0.3 M in the case of a disaccharide; preferably 0.1 M to 0.3 M in the case of a trisaccharide.
  • the collagen in the sustained-release pharmaceutical composition of the present invention may be any collagen as long as it can undergo gelation under physiological conditions.
  • the collagen is distinguished from gelatin as a denatured product of the collagen.
  • the collagen has a triple-helical structure formed of an assembly of three helical polypeptide chains, whereas gelatin does not have such structure.
  • the triple-helical structure in the collagen is formed of molecules each including a repeating sequence of (Gly-X-Y) n (X and Y each represent any amino acid) .
  • the molecular weight of the collagen in the pharmaceutical composition of the present invention is 1 to 2,000 kDa, preferably 10 to 1,000 kDa, more preferably 100 to 500 kDa.
  • the molecular weight of the collagen is less than 1 kDa, for example, there is a problem in that the gelation of the collagen does not occur under physiological conditions.
  • the molecular weight is more than 2,000 kDa, for example, there is a problem in that the gelation occurs even under conditions other than the physiological conditions, which makes it difficult to handle the formulation.
  • the molecular weight as used herein means a total molecular weight, and is generally measured by a GPC method or the like.
  • atelocollagen obtained by treatment with a protease such as pepsin ( Stenzel et al., Collagen as a biomaterial, Annual Review of Biophysics and Bioengineering, (1974) 3, 231 ) as well as various types of natural collagens.
  • atelocollagen there may be used atelocollagen derived from any of various living beings such as a chicken, cattle, a pig, and a human.
  • a chemically modified collagen a collagen-like molecule in which synthetic polypeptides each including a repeating sequence of Gly-X-Y (X and Y each represent any amino acid) such as (Pro-Hyp-Gly) 5 form a triple helix, or the like may be used as long as it can undergo gelation under physiological conditions.
  • synthetic polypeptides each including a repeating sequence of Gly-X-Y (X and Y each represent any amino acid) such as (Pro-Hyp-Gly) 5 form a triple helix, or the like
  • gelatin or a collagen peptide does not undergo gelation under physiological conditions, and hence is not preferred as the collagen to be used in the sustained-release pharmaceutical composition of the present invention.
  • the concentration of the collagen in the present invention may be any concentration as long as it is such a concentration that gelation occurs under physiological conditions at the final concentration in the pharmaceutical composition.
  • the concentration of the collagen is 0.01 to 30% by weight (% [w/v]), preferably 0.05 to 20% by weight (% [w/v]), more preferably 0.1 to 10% by weight (% [w/v]).
  • concentration of the collagen is less than 0.01% by weight (% [w/v]), for example, there is a problem in that the gelation of the collagen does not occur.
  • the concentration is more than 30% by weight (% [w/v]), for example, there is a problem in that the sustained-release pharmaceutical composition of the present invention cannot be handled as a solution.
  • the blending ratio of the collagen to the sugar in the sustained-release pharmaceutical composition of the present invention has only to be such a blending ratio that the sugar is capable of inhibiting the gelation of the collagen.
  • the weight (g) of the sugar with respect to 1 part by weight (1 g) of the collagen has only to be 1:0.005 to 1:5,000, and has only to be preferably 1:0.01 to 1:1,000, more preferably 1:0.02 to 1:500, still more preferably 1:0.1 to 1:100.
  • the weight (g) of the sugar with respect to 1 part by weight (1 g) of the collagen is less than 0.005, for example, there is a problem in that an effect of inhibiting gelation is not obtained.
  • the weight is more than 5,000, for example, there is a problem in that the gelation of the collagen is completely inhibited, and thus the composition does not exhibit drug sustained-release property. Further, it should be noted that the gelation may be inhibited at a higher level by increasing the weight (g) of the sugar with respect to 1 part by weight (1 g) of the collagen, to thereby produce a pharmaceutical composition having a high proportion of immediate-release property.
  • the collagen is present as a collagen solution by being dissolved in a medium.
  • a medium may be used as the medium as long as it satisfies a pH and a salt concentration under physiological conditions.
  • a medium such as purified water for injection, physiological saline, or a buffer (e.g., a physiological phosphate buffer, an acetate buffer, or a citrate buffer).
  • the sustained-release pharmaceutical composition of the present invention contains a drug as an active ingredient.
  • the drug has only to be a physiologically active substance having an action of preventing and/or treating a disease or a pathologic condition.
  • the drug include physiologically active substances having actions of promoting tissue regeneration and of promoting and enhancing a physiological action, and ones used as general pharmaceuticals such as an anti-cancer agent and an antibiotic.
  • the drug may be any of a protein, a peptide, a glycoprotein, a polysaccharide, a nucleic acid, and a low-molecular-weight compound. Of those, a protein, a peptide, or a glycoprotein is preferred.
  • the peptide is a molecule in which two or more amino acids are bound together, and the protein and the peptide have substantially the same meaning.
  • the glycoprotein has a sugar chain bound to a protein.
  • the protein as the drug in the present invention may be any protein, and for example, one having a molecular weight of 1 to 1,000 kDa may be used.
  • fibroblast growth factor FGF
  • EGF epidermal growth factor
  • NGF nerve growth factor
  • IGF insulin-like growth factor
  • CSF colony stimulating factor
  • G-CSF granulocyte-colony stimulating factor
  • TGF transforming growth factor
  • VEGF vascular endothelial growth factor
  • GH growth hormone
  • EPO erythropoietin
  • HGF hepatocyte growth factor
  • TNF- ⁇ tumor necrosis factor
  • TNF- ⁇ lymphotoxin
  • interleukin an interferon, insulin, parathyroid hormone (PTH), a platelet-derived growth factor (PDGF), a vaccine antigen, an antibody, lysozyme (cell wall digesting enzyme), serrapeptase (anti-inflammatory enzyme preparation), and Pronase (anti-inflammatory enzyme preparation).
  • the FGF is preferably basic fibroblast growth factor (FGF), an epidermal growth factor (EGF), a nerve growth factor (NGF), an insulin-like growth factor (
  • nucleic acid examples include: a gene encoding an enzyme, a hormone, a cytokine, a colony stimulating factor, a coagulation factor, a regulatory protein, a transcription factor, a receptor, or a structural protein; and an antisense oligonucleotide, siRNA, miRNA, RNA, DNA, or aptamer targeting such gene and formed of a single-stranded or double-stranded nucleic acid formed of an oligonucleotide or an oligonucleoside.
  • the concentration of the drug in the sustained-release pharmaceutical composition of the present invention has only to be one sufficient to exhibit an action of preventing and/or treating a disease or a pathological condition in vivo of a subject (patient) to which the sustained-release pharmaceutical composition of the present invention is administered.
  • the concentration of the drug has only to be comparable to the effective concentration of a pharmaceutical to be generally used. Further, the concentration may be a concentration at which it has been clarified that effectiveness is exhibited by the drug only. However, a case where the mixing with the collagen decreases or increases the concentration at which the effectiveness is exhibited as compared to the case of the drug only is also envisaged.
  • the sustained-release pharmaceutical composition of the present invention is preferably topically used, and hence the concentration of the drug in the sustained-release pharmaceutical composition has only to be determined focusing on the local concentration of the drug.
  • the sustained-release pharmaceutical composition of the present invention may further contain a pharmaceutically acceptable carrier.
  • a sustained-release pharmaceutical composition of the present invention is an injectable injection, for example, a dispersant, a surfactant, a tonicity agent, a pH adjusting agent, a soothing agent, a stabilizer, a preservative, or a colorant may be used as the pharmaceutically acceptable carrier as necessary.
  • the sustained-release pharmaceutical composition of the present invention is preferably used parenterally as an injection.
  • the administration of the composition may be intradermal administration, subcutaneous administration, intramuscular administration, intracavity administration, or administration to an organ or a tissue.
  • the sustained-release pharmaceutical composition of the present invention may be administered to vertebrates (such as: fishes; amphibians; reptiles; birds including a chicken and the like; and mammals including a mouse, a rat, a rabbit, a cat, a dog, cattle, a pig, a horse, a goat, a sheep, a monkey, a human, and the like) as subjects.
  • the sustained-release pharmaceutical composition of the present invention may be used as an angiogenesis regulator.
  • the angiogenesis regulation means an action of promoting and/or inhibiting angiogenesis.
  • physiologically active substance exhibiting an angiogenesis regulating action examples include FGF (preferably bFGF), PDGF, VEGF, HGF, G-CSF, fumagillin, bevacizumab, ranibizumab, a recombinant human monoclonal Fab antibody that neutralizes active forms of vascular endothelial growth factor A (VEGF-A)), cimetidine, celecoxib), azaspirene, and pegaptanib (nucleic acid pharmaceutical).
  • FGF preferably bFGF
  • PDGF vascular endothelial growth factor A
  • VEGF-A vascular endothelial growth factor A
  • VEGF-A vascular endothelial growth factor A
  • cimetidine cimetidine
  • celecoxib celecoxib
  • azaspirene azaspirene
  • pegaptanib pegaptanib
  • the angiogenesis regulator of the present invention When the angiogenesis regulator of the present invention is administered to a living body, the local concentration of the physiologically active substance exhibiting an angiogenesis regulating action as an active ingredient increases early and the substance can be released in a sustained manner over a long period and kept at an appropriate concentration.
  • the angiogenesis regulator may be used for various treatment applications using an angiogenesis regulating action.
  • the physiologically active substance exhibiting an angiogenesis regulating action is bFGF
  • the substance is released in an amount sufficient to provide a subject with an initial loading dose of bFGF within a time of 2 hours or less, 1 hour or less, 30 minutes or less, or about 10 minutes or less. It is meant that the angiogenesis regulator is released in an amount sufficient to provide a subject with a continuous maintenance dose of bFGF for a time of 12 hours or more, 16 hours or more, or 20 hours or more and up to about 24 hours, up to about 28 hours, or up to about 32 hours after administration.
  • the angiogenesis regulator can be prepared so that the final concentration of bFGF is 25 ⁇ g/mL, to thereby set the dosage (dose) of bFGF at the local site of a subject to about 100 to about 500 ⁇ g, preferably about 200 ⁇ g.
  • the angiogenesis regulator of the present invention may be applied to, for example, angiogenesis promotion, angiogenesis inhibition, tissue regeneration, wound healing, bone disease treatment, periodontal disease treatment, cartilage disease treatment, or cancer treatment.
  • the angiogenesis regulator is preferably used for angiogenesis promotion or tissue regeneration.
  • vascular occulsive diseases and ischemic diseases concurrent to the vascular occlusion in general.
  • the disease is any of chronic arterial occlusive diseases in general and examples thereof include arteriosclerosis obliterans, Buerger's disease, diabetic gangrene, myocardial infarction, and angina pectoris.
  • the dosage of the angiogenesis regulator of the present invention may be appropriately adjusted depending on, for example, a disease to be treated and the age and body weight of a patient.
  • the dosage in terms of bFGF is generally selected from the range of about 0.01 to about 500 ⁇ g, preferably the range of about 1 to about 300 ⁇ g, per adult human patient, and can be injected into an affected site or a peripheral site thereof. Further, when the effect in single administration is insufficient, the administration may also be performed a plurality of times.
  • the sustained-release pharmaceutical composition of the present invention may be produced by mixing ingredients including a drug, a collagen, and a sugar at predetermined concentrations and blending ratios.
  • the blending order of the drug, the collagen, and the sugar is not particularly limited, and for example, the composition may be produced by blending the ingredients in any of the following orders.
  • a drug release rate may be controlled more freely by appropriately adjusting the concentrations, the blending ratios, and the blending order.
  • the present invention also encompasses a sustained-release base, including: a collagen; and at least one kind of sugar selected from disaccharides and trisaccharides wherein the concentration of the sugar is 0.1 M to 0.5 M, and wherein the sustained-release base is used for manufacture of a sustained-release pharmaceutical composition comprising the collagen, the sugar and a drug.
  • the sustained-release base means a solvent or base for the sustained-release pharmaceutical composition.
  • the sustained-release pharmaceutical composition may be produced by adding a drug as an active ingredient to the sustained-release base of the present invention, followed by mixing.
  • At least one kind of sugar selected from disaccharides and trisaccharides can be allowed to be present in a pharmaceutical composition containing a drug and a collagen, to thereby impart immediate-release property and sustained-release property to the pharmaceutical composition.
  • a method of controlling a drug release rate is also disclosed.
  • a blending operation is preferably performed at a temperature at which the collagen is not denatured, preferably at a low temperature of 2 to 10°C, and in a state in which the sugar is present, a mixing operation may be performed at 0 to 50°C, more preferably 10 to 40°C.
  • sucrose solution was added to a collagen solution (pH 7.4, final concentration: 3% [w/v]) (product name: KOKEN ATELOCOLLAGEN IMPLANT) so that the final concentration was 0, 0.01, or 0.25 M, followed by mixing.
  • the mixed solution was measured for its time-dependent change in absorbance at 400 nm with a spectrophotometer while being heated at 37°C.
  • FIG. 1 shows the results.
  • “a” shows the result in the case of a collagen only
  • “b” shows the result in the case of a blend of a collagen and 0.01 M sucrose
  • “c” shows the result in the case of a blend of a collagen and 0.25 M sucrose.
  • the collagen solution to which no sucrose was added underwent gelation in about 10 minutes. In each of the collagen solutions to which sucrose was added, its gelation was delayed in a sucrose concentration-dependent manner.
  • Lysozyme was dissolved in a physiological buffer (PBS, pH 7.4) containing sucrose (final concentration: 0.05 M) or a physiological buffer containing no sucrose. After that, the solution and an atelocollagen solution (final concentration: 3% [w/v]) (product name: KOKEN ATELOCOLLAGEN IMPLANT) were mixed and formulated.
  • the resultant formulation was gently added to a solution containing PBS as an external liquid in a test tube, followed by heating at 37°C, and the amount of the lysozyme released into the external liquid in a time-dependent manner was measured by HPLC (system integrator: C-R7A [Shimadzu Corporation], system controller: SCL-6B [Shimadzu Corporation], pump: LC-9A [Shimadzu Corporation], degasser: ERC-3522 [Elma], detector: SPC-6AV [Shimadzu Corporation], column: Cosmosil C18-AR [NACALAI TESQUE, INC.]).
  • HPLC system integrator: C-R7A [Shimadzu Corporation]
  • system controller SCL-6B [Shimadzu Corporation]
  • pump LC-9A [Shimadzu Corporation]
  • degasser ERC-3522 [Elma]
  • detector SPC-6AV [Shimadzu Corporation]
  • column Cosmosil C18-AR [NACAL
  • FIG. 2 shows the results.
  • the open circle shows the result in the case where sucrose is absent
  • the filled circle shows the result in the case where sucrose is present.
  • the amount of the lysozyme released into the external liquid was found to be large in the case of containing sucrose as compared to the case of containing no sucrose.
  • lysozyme (MW: 14 kDa) (final concentration: 2 mg/mL); sucrose (final concentration: 0.25 M); and atelocollagen (final concentration: 3% [w/v]) (product name: KOKEN ATELOCOLLAGEN IMPLANT).
  • mice 100 ⁇ L of each of the formulations according to the above-mentioned items a) to c) were administered to the dorsal subcutis of mice (DDY, female, 20-week-old) .
  • the presence or absence of gelation of atelocollagen in vivo was confirmed 24 hours after the administration to the mice.
  • FIGS. 3 show the states of the respective formulations in vivo after 24 hours.
  • FIGS. 3a to 3c show the results of the respective formulations according to the above-mentioned items a) to c), and
  • FIG. 3d shows the result of the control formulation.
  • Each of the formulations according to the above-mentioned items a) to c) and the control formulation formed a white gel in the dorsal subcutis of the mice.
  • lysozyme MW: 14 kDa
  • sucrose sucrose
  • atelocollagen product name: KOKEN ATELOCOLLAGEN IMPLANT
  • Each of the formulations was prepared so that the final concentrations of lysozyme, sucrose, and atelocollagen were 5 mg/mL, 0.25 M, and 3% [w/v], respectively.
  • FIG. 4 shows the results.
  • the release amount of the lysozyme varied depending on a change in mixing procedure.
  • a drug as any of various proteins shown in Table 1 below
  • sucrose final concentration 0.25 M
  • atelocollagen final concentration: 3% [w/v]
  • the formulation was performed by: dissolving sucrose in a physiological buffer; mixing the solution with an atelocollagen solution; and adding a protein dissolved in a physiological buffer.
  • FIGS. 5 show the states of the respective formulations in vivo after 24 hours.
  • FIG. 5a shows the result of transferrin
  • FIG. 5b shows the result of phosphorylase b
  • FIG. 5c shows the result of myosin heavy chain
  • FIG. 5d shows the result of ferritin type I
  • FIG. 5e shows the result of thyrogloblin.
  • Each of the formulations formed a gel in the dorsal subcutis of the mice.
  • bFGF MW: 17 kDa
  • sucrose final concentration: 0.25 M
  • atelocollagen final concentration: 3% [w/v]
  • product name: KOKEN ATELOCOLLAGEN IMPLANT product name: KOKEN ATELOCOLLAGEN IMPLANT
  • FIGS. 6 show the results.
  • the periphery of the blood vessel at the administration site was found to become reddish (arrows), suggesting capillary angiogenesis.
  • FIG. 6b In the case of administering a formulation containing no sucrose ( FIG. 6b ), no clear redness was observed in the periphery of the administration site (arrows), suggesting that angiogenesis was delayed as compared to the formulation containing sucrose.
  • no capillary angiogenesis was observed as with the formulation containing no sucrose ( FIG. 6c ).
  • Such difference in effect of bFGF in the subcutis difference between FIGS.
  • a collagen solution (final concentration: 3% [w/v]) (product name: KOKEN ATELOCOLLAGEN IMPLANT (KOKEN CO., LTD.)) was added to a solution of bFGF containing white soft sugar (product name: FIBLAST Spray (KAKEN PHARMACEUTICAL CO., LTD.)) to prepare a formulation containing 1.0 mg/mL bFGF.
  • mice Under 0.5 to 4% isoflurane anesthesia, the mice were fixed in the dorsal position, the thigh of the right hindlimb was incised, and the femoral artery on the central and peripheral sides and the saphenous artery on the periphery side were each ligated with a suture to block the blood circulation. A penicillin G potassium solution was dropped and the incised site was sutured.
  • the formulation was administered with an injector to five sites of the muscle of the right ischemic hindlimb in an amount of 20 ⁇ L per site so that the dosage was 100 ⁇ g of bFGF per animal.
  • the recovery of blood flow at the hindlimb ischemic site was evaluated through use of a laser doppler blood flow imager (OZ-1, OMEGAWAVE, INC.) 4 weeks after the administration of the formulation.
  • OZ-1 laser doppler blood flow imager
  • mice Under 0.5 to 4% isoflurane anesthesia, the mice were fixed in the dorsal position, and blood flow rates were measured at the right hindlimb (ischemic limb) and the left hindlimb (normal limb) to calculate a blood flow rate of ischemic limb/normal limb.
  • FIG. 7 shows the results.
  • the blood flow rate was significantly recovered in a group in which the formulation containing sucrose was administered as compared to a group in which the collagen was administered as a control.
  • phase I and II clinical trials were performed as described below.
  • Subjects were cases who were diagnosed as negative for an intradermal reaction for bFGF-containing atelocollagen (AC) after giving informed consent out of patients with Fontaine stages III and IV who had lower extremity peripheral vascular disease with pain at rest or ischemic ulcer and necrosis and who did not show any improvement in clinical symptom even after subjected to any medical and surgical treatment. No age limit was set.
  • AC bFGF-containing atelocollagen
  • Patients excluded from the trials were patients who had experienced a lower extremity revascularization procedure (surgical bypass procedure, PTA) within 3 months, patients who had severe cardiac dysfunctions such as angina pectoris, myocardial infarction, and congestive heart failure, patients who had collagen disease, malignant neoplasm, infectious diseases, and diabetic retinopathy, pregnant women, and patients who were diagnosed as positive in an intradermal reaction for an atelocollagen solution.
  • surgical bypass procedure PTA
  • Table 2 shows the patients as the subjects of this example. [Table 2] Cases Age Gender Fontaine Disease 1 41 Male III Buerger's disease 2 87 Female III Peripheral arterial disease
  • Buerger's disease (Synonym: Thromboangiitis obliterans): TAO Peripheral arterial disease: PAD
  • atelocollagen (AC) (product name: KOKEN ATELOCOLLAGEN IMPLANT (KOKEN CO., LTD.)) containing 200 ⁇ g of bFGF containing white soft sugar (product name: FIBLAST Spray (KAKEN PHARMACEUTICAL CO., LTD.)) was injected into the gastrocnemius muscle of ischemic lower extremity (100 ⁇ l ⁇ 40 sites). No placebo or untreated group was set.
  • the atelocollagen solution containing 200 ⁇ g of bFGF was evaluated for its safety and therapeutic effect.
  • Primary endpoint the safety was confirmed as the primary endpoint.
  • Secondary endpoint Evaluations were performed from the two viewpoints of primary outcome assessment and secondary outcome assessment as the secondary endpoint.
  • VAS visual analogue scale
  • a distance that a patient was able to walk in 6 minutes was measured in the corridor of a hospital ward.
  • the index was calculated from the ratio of a brachial blood pressure to an ankle blood pressure (brachial blood pressure/ankle blood pressure).
  • the skin was heated to 43.5°C in advance, and the measurement was performed by attaching a probe to the frontal skin about 10 cm away from the tibial tuberosity toward the periphery side.
  • FIGS. 8 show the results. Both the patients shown in Table 2 did not show any change during a hospitalization period of 4 days, in systemic symptom, blood, and urine examination, and were discharged from the hospital on schedule. No adverse event due to the administration of the formulation was found. In each of the patients, the pain at rest was improved in a time-dependent manner after the administration of the formulation as compared to before the treatment. With regard to the ABI, an improving effect was found in the patient with Buerger's disease showing a low value for the index. With regardto the 6-minute walk distance, animproving effect was found as compared to before the treatment in each of the patients . With regard to each of TcO 2 and SPP, an improving effect was found after the administration of the formulation.
  • Distilled water (OTSUKA DISTILLED WATER, Cat. 1324, Lot. 0J90N), 2.5 M sucrose (Wako Pure Chemical Industries, Ltd., Cat. 196-00015, Lot. CKM1674), 100 mg/mL lysozyme (Tokyo Chemical Industry Co., Ltd., Cat. L072), and 10 ⁇ PBS (DULBECCO'PBS, Dainippon Sumitomo Pharma Co., Ltd., Cat. 28-103-05 FN, Lot. 896458) were added to each of the various collagen solutions at 0.2% [w/v] to prepare a solution having final concentrations of 0.2% [w/v] collagen/0.25 M sucrose/10 mg/mL lysozyme/1 ⁇ PBS. At this time, the ingredients were added so that the collagen, distilled water, sucrose, lysozyme, and PBS were mixed in this order.
  • FIGS. 9 shows the results.
  • “a” shows the result of a formulation containing no lysozyme and no sucrose and containing a collagen only at 0.1% [w/v]
  • "b” shows the result of a formulation containing no lysozyme and containing 0.25 M sucrose and a collagen at 0.1% [w/v]
  • "c” shows the result of a formulation containing 10 mg/mL lysozyme, 0.25 M sucrose, and a collagen at 0.1% [w/v].
  • the results revealed that the addition of the sugar suppressed gelation in various collagen solutions containing type I, II, and III atelocollagens, type I native collagen, and shark atelocollagen.
  • FIG. 11 shows the results.
  • "a” shows the result of a formulation containing no stachyose
  • "b” shows the result of a formulation containing stachyose.
  • the fibrillogenesis time of the collagen solution was delayed.
  • the tetrasaccharide suppressed the fibrillogenesis of the collagen solution.
  • the tetrasaccharide is considered to have a low fibrillogenesis suppressing effect as compared to the monosaccharide, the disaccharide, and the trisaccharide.
  • the kind and concentration of a sugar to be added were changed, and an ability of the sugar to suppress the gelation of a collagen solution was confirmed by measuring a fibrillogenesis time.
  • the collagen was prepared so that the final concentration was 3% [w/v].
  • KAI pH 7, PB buffer
  • the kinds and final concentrations of the used sugars are as described below.
  • FIGS. 12 show the results.
  • "a” shows the result of a formulation containing no sugar
  • "b” shows the result of a formulation containing a sugar at 0.05 M
  • "c” shows the result of a formulation containing a sugar at 0.125 M.
  • Maltotetraose exhibited a fibrillogenesis suppressing effect comparable to that of the monosaccharide.
  • substantially no delay of the fibrillogenesis time was able to be confirmed in the 3% [w/v] collagen solution.
  • a collagen solution was prepared so that the final concentration was 3% [w/v] or 0.4% [w/v].
  • Methods for the preparation were as follows: collagen solutions having final concentrations of 3% [w/v] and 0.4% [w/v] were produced by the same methods as those of Reference Example 3(1) and Reference Example 3(2), respectively.
  • the kinds of the used sugars were glucose, sucrose, raffinose, and stachyose (the same as in those of Reference Examples 2 and 3), and the final concentration was 0.05 M.
  • Each of the prepared solutions was measured for its time-dependent change in absorbance at 400 nm with a spectrophotometer while being heated at 37°C.
  • FIGS. 13 show the results.
  • “a” shows the result of a formulation containing a collagen at 3% [w/v]
  • “b” shows the result of a formulation containing a collagen at 0.4% [w/v].
  • Sugars including a monosaccharide to a pentasaccharide constructed only of glucose were used as sugars to be added, and the ability of each of the sugars to suppress the gelation of a collagen solution was confirmed by measuring a fibrillogenesis time.
  • the collagen solution was prepared so that the final concentration was 2% [w/v].
  • KOKEN ATELOCOLLAGEN IMPLANT [KOKEN CO., LTD.] was used as the collagen.
  • the used sugars were glucose [Tokyo Chemical Industry Co., Ltd.], maltose [Wako Pure Chemical Industries, Ltd.], maltotriose [Wako Pure Chemical Industries, Ltd.], maltotetraose [Tokyo Chemical Industry Co., Ltd.], and maltopentaose [Wako Pure Chemical Industries, Ltd.].
  • the various sugars were dissolved in physiological saline [Otsuka Pharmaceutical Co., Ltd.] to prepare 1.25 M sugar solutions.
  • the collagen (final concentration: 2% [w/v]) and the various sugars (final concentration: 0.3 M) were filled into syringes, which were connected in series via a connector, followed by thorough mixing by reciprocation 50 times on ice.
  • the contents were transferred to a 1.5-mL sampling tube, subjected to centrifugal defoaming (13,000 rpm, 4°C), and then heated in a thermostat bath (37°C). The state of the gelation was observed after 0 minutes, 10 minutes, 30 minutes, 60 minutes, 12 hours, 24 hours, and 3 days.
  • FIG. 14 shows the results.
  • Each of the monosaccharide, the disaccharide, and the trisaccharide had a large gelation suppressing effect, and the effect became the maximum in the disaccharide or the trisaccharide.
  • the gelation suppressing effect became lower.
  • a possible mechanism through which the gelation suppressing effect becomes lower is that a physical or charge factor reduces the effect.
  • sucrose final concentration: 0.25 M
  • lysozyme final concentration: 10 mg/mL
  • atelocollagen final concentration: 0.05, 0.1, 0.5, or 1% [w/v]
  • the formulation was performed by mixing sucrose with an atelocollagen solution and then adding lysozyme.
  • FIGS. 15 show the results.
  • the numerical value described on the left of each photograph represents the concentration of the collagen in the formulation.
  • Gelation was confirmed in the formulations containing the collagen at 0.1% [w/v] and 0.05% [w/v] and the formulations containing the collagen at 0.5% [w/v] and 1% [w/v]. It was able to be confirmed that the formulations containing the collagen at concentrations of 0.05 to 1% [w/v] (containing 0.25 M sucrose and 10 mg/mL lysozyme) were capable of undergoing gelation 24 hours after the subcutaneous implantation in the mice.
  • the state of the gelation in the subcutis of the mice after 24 hours in the case of changing the kind of a sugar to be mixed was observed.
  • the kinds and concentrations of the sugars are as shown in Table 3 below.
  • [Table 3] Kinds and final concentrations of used sugars Study group Reference numeral in FIGS. 16
  • Kind and final concentration of sugar Comparison among kinds of sugars a 0.05 M Glucose b 0.05 M Sucrose c 0.05 M Raffinose d 0.05 M Stachyose Comparison among monosaccharides e 0.5 M Glucose f 0.25 M Glucose g 0.5 M Fructose h 0.25 M Fructose Comparison between disaccharides i 0.25 M Sucrose j 0.25 M Maltose
  • any of the various sugars shown in Table 3 any of the various sugars shown in Table 3; lysozyme (final concentration: 10 mg/mL); and atelocollagen (final concentration: 3% [w/v]) (KOKEN ATELOCOLLAGEN IMPLANT [KOKEN CO., LTD.]).
  • the formulation was performed by dissolving sucrose in a physiological saline solution, mixing the solution with an atelocollagen solution, and adding lysozyme dissolved in a physiological saline solution. 50 ⁇ L of each of the formulations containing the sugars shown in Table 3 were administered to one site of the dorsal subcutis of mice (ICR, female, 9-week-old), and the state of the collagen solution 24 hours after the administration was observed.
  • FIGS. 16 show the results.
  • FIGS. 16a to 16j show the results of the formulations produced by using the kinds and sugar concentrations of sugars corresponding to the reference numerals in Table 3 above, respectively. Irrespective of the kinds and concentrations of the sugars, gelation was able to be confirmed in each of the formulations.
  • sucrose final concentration: 0.25 M
  • lysozyme final concentration: 10 mg/mL
  • atelocollagen final concentration: 0.05, 0.1, 0.5, or 1% [w/v]
  • the formulation was performed by mixing sucrose with an atelocollagen solution and then adding lysozyme. 100 ⁇ L of each of the formulations were administered to one site of the dorsal subcutis of mice (ICR, female, 9-week-old), and the state of the collagen solution was observed 24 hours and 48 hours after the administration.
  • the formulation of the present invention contains sucrose at 0.25 M, and hence is expected to undergo gelation through the release of the sugar and the fibrillogenesis of atelocollagen after the absorption of the moisture of a body fluid based on an osmotic pressure .
  • the sample having a concentration below a given one is predicted to have a low long-term retention property.
  • the following sugars were used as sugars to be added, and an ability of the sugar to suppress the gelation of a collagen solution was confirmed by measuring a fibrillogenesis time.
  • Each of various sugar solutions was added to a collagen solution (pH 7.4, final concentration: 3% [w/v]) (KOKEN ATELOCOLLAGEN IMPLANT [KOKEN CO., LTD.]) so that the final concentration was 0.01 M, followed by mixing.
  • the mixed solution was measured for its time-dependent change in absorbance at 400 nm with a spectrophotometer while being heated at 37°C.
  • FIG. 18 shows the results.
  • "a” shows the result of a formulation containing no sugar
  • "b” shows the result of a formulation containing sucrose
  • "c” shows the result of a formulation containing glucose
  • “d” shows the result of a formulation containing glucuronic acid.
  • sucrose and glucose has a high gelation suppressing effect.
  • glucuronic acid has a low gelation suppressing effect as compared to glucose.
  • the formulation of the present invention containing a high molecular weight protein (thyroglobulin (669 kDa)) as a drug was produced, and its gelation 24 hours after subcutaneous implantation in mice was observed.
  • the sustained-release amount of thyroglobulin was quantitatively measured by HPLC in a time-dependent manner.
  • the formulation of the present invention was produced by adjusting the final concentrations of atelocollagen, sucrose, and the protein to 3% [w/v], 0.25 M, and 10 mg, respectively. 3.5% [w/v] atelocollagen Lot. 1-175, Kasai Lac, 2.5 M sucrose, and a 100-mg protein solution were mixed at a blending ratio of 8:1:1. First, syringes filled with atelocollagen and sucrose were connected in series on ice, followed by thorough mixing by reciprocation 50 times . The mixed liquid was mixed with the protein solution by syringe reciprocation 50 times immediately before subcutaneous injection into mice.
  • mice were purchased from Japan SLC, Inc. ICR strain female mice were purchased at 7-week-old 1 week before use and used at 8-week old.
  • Each of samples after 0, 10 minutes, 30 minutes, 45 minutes, 1 hour, 3 hours, 6 hours, and 12 hours was extirpated and heated to 80°C in an Eppendorf tube so as to denature the collagen into a solution form.
  • Each of the samples was analyzed through use of Agilent HPLC and an SEC-4 gel filtration column to quantify the concentration of the protein in the gel. In the HPLC, thyroglobulin is eluted at 5.927 minutes.
  • FIGS. 19 to 21 show the results. Until 3 hours after the injection, fast release was observed. After that, the rate was turned into a low sustained-release rate. This phenomenon was considered to have a close relationship with in vivo gelation. Irrespective of the molecular weight of a protein to be mixed, the release rate of the protein exhibited biphasic property of fast release in the initial period and slow release after gelation. A high release rate in the initial period and a time that elapses before gelation may be changed by changing the concentration of sucrose to be mixed.
  • the drug as an active ingredient can be released in a sustained manner in an embodiment mode using immediately effective release and slow sustained release in combination in vivo, and the drug release rate can be controlled.
  • the composition is useful.
  • the drug release rate can be controlled. Hence, there is a possibility that the treatment or the like of even an indication on which no effectiveness has been found hitherto can be performed.
  • the sustained-release pharmaceutical composition of the present invention when a sugar is added to the collagen, its gelation is inhibited, and the composition can be prepared at room temperature, which can facilitate operations. Further, a drug, which has not been applicable to a preparation operation at low temperature, can be used. Thus, the composition is useful.

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EP11832614.9A 2010-10-15 2011-10-14 Sustained-release pharmaceutical composition Active EP2628488B1 (en)

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JP2010232113 2010-10-15
PCT/JP2011/073632 WO2012050184A1 (ja) 2010-10-15 2011-10-14 徐放性医薬組成物

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EP2628488A1 EP2628488A1 (en) 2013-08-21
EP2628488A4 EP2628488A4 (en) 2016-07-27
EP2628488B1 true EP2628488B1 (en) 2020-04-08

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JP (1) JP5860406B2 (ja)
KR (1) KR20140015264A (ja)
CN (1) CN103221066A (ja)
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WO (1) WO2012050184A1 (ja)

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US9795714B2 (en) * 2013-12-24 2017-10-24 Ares Trading S.A. FGF-18 formulation in alginate/collagen hydrogels

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JPS56122317A (en) 1980-02-29 1981-09-25 Koken:Kk Drug transporting material and its preparation
JPH0759522B2 (ja) 1985-12-27 1995-06-28 住友製薬株式会社 徐放性製剤の製造法
JPH0543453A (ja) 1991-08-20 1993-02-23 Sumitomo Pharmaceut Co Ltd 創傷治癒促進用局所用徐放性製剤
IL105529A0 (en) * 1992-05-01 1993-08-18 Amgen Inc Collagen-containing sponges as drug delivery for proteins
JPH10167987A (ja) 1996-10-09 1998-06-23 Sumitomo Pharmaceut Co Ltd 徐放性製剤
KR100600464B1 (ko) * 1998-05-22 2006-07-13 다이닛본 스미토모 세이야꾸 가부시끼가이샤 안정한 유전자 제제
WO2002040072A2 (en) * 2000-11-15 2002-05-23 Bio Syntech Canada Inc. Filler composition for soft tissue augmentation and reconstructive surgery
US20040047892A1 (en) 2000-11-15 2004-03-11 Desrosiers Eric Andre Filler composition for soft tissue augmentation and reconstructive surgery
JPWO2008120741A1 (ja) 2007-03-30 2010-07-15 国立大学法人 千葉大学 塩基性線維芽細胞増殖因子の徐放性製剤
WO2009119073A1 (ja) * 2008-03-28 2009-10-01 北海道公立大学法人札幌医科大学 皮膚の老化や瘢痕の治療剤
JP2010232113A (ja) 2009-03-30 2010-10-14 Otax Co Ltd Ｒｆ−ｍｅｍｓスイッチ、ｒｆ−ｍｅｍｓスイッチの製造方法、アンテナ切り替え装置、携帯電話機、携帯用情報端末機器、ｉｃテスト用機器

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KR20140015264A (ko) 2014-02-06
CN103221066A (zh) 2013-07-24
JP5860406B2 (ja) 2016-02-16
JPWO2012050184A1 (ja) 2014-02-24
EP2628488A1 (en) 2013-08-21
US20130225492A1 (en) 2013-08-29
US9301917B2 (en) 2016-04-05
SG189402A1 (en) 2013-05-31
EP2628488A4 (en) 2016-07-27
WO2012050184A1 (ja) 2012-04-19

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